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Cell Signaling Technology Inc cleaved notch 1

The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of <t>Notch</t> <t>1</t> (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Cleaved Notch 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 954 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin"

Article Title: Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

Journal: JID Innovations

doi: 10.1016/j.xjidi.2025.100444

Figure Legend Snippet: The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of Notch 1 (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Techniques Used: Inhibition, Activity Assay, Fluorescence, Labeling, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Control, High Content Screening

Membrane:

Article Title: Whole Exome Sequencing of Lacrimal Gland Adenoid Cystic Carcinoma
Article Snippet: Whole cell lysates from transfected U2OS cells were run on a 4% to 20% SDS-PAGE and transferred to polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). .. The membrane was incubated with anti-cleaved Notch 1 antibody (cat # 4147; Cell Signaling Technology, Danvers, MA, USA) and visualized with a Western blotting substrate (Pierce ECL; Thermo Fisher Scientific). .. The membrane was then stripped and reprobed with anti-GAPDH antibody (cat# sc-47724; Santa Cruz Biotechnology, Dallas, TX).

Incubation:

Western Blot:

Image Search Results

Journal: JID Innovations

Article Title: Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

doi: 10.1016/j.xjidi.2025.100444

Figure Lengend Snippet: The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of Notch 1 (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Article Snippet: Membranes were blocked with 10% nonfat dry milk for 1 hour and incubated with primary antibodies against NOTCH 1 (number 3608, Cell Signaling Technology), cleaved NOTCH-1 (number 4147, Cell Signaling Technology), c-MYC, or β-actin (ab8227, Abcam) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Fluorescence, Labeling, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Control, High Content Screening

Fig. 1 A Notch signature is present in MBMs. A-B GSE200218: (A) Uniform Manifold Approximation and Projection (UMAP) showing the combined tumor cell cluster of melanoma brain (n = 15) and peripheral (n = 10) metastases (top), with Notch1 signature distribution among the two (bottom). B Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) in brain tumor cell cluster compared to the peripheral metastases. C Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) in GSE200217 in MBM compared to extracranial metastases. D Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) for GSE50496

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 1 A Notch signature is present in MBMs. A-B GSE200218: (A) Uniform Manifold Approximation and Projection (UMAP) showing the combined tumor cell cluster of melanoma brain (n = 15) and peripheral (n = 10) metastases (top), with Notch1 signature distribution among the two (bottom). B Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) in brain tumor cell cluster compared to the peripheral metastases. C Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) in GSE200217 in MBM compared to extracranial metastases. D Bubble plot depicting normalized enrichment score (NES) for several Hallmark gene sets from the molecular signature databases (MSigDB) for GSE50496

Article Snippet: Antibodies used were: anti cleaved-Notch1 (Novus biologicals, NB100-78486), Histone H2 A.X (clone D17 A3 XP, Cell signaling Technology, cat #7631); Histone H3 (clone D1H2, Cell signaling Technology, Cat #4499); Histone H2B, (Proteintech cat #15,857–1-AP); Histone H4 (Proteintech cat # 16,047–1-AP), anti-phospho-ChK1S345 (clone 133D3, Cell signaling Technology, cat #2348), anti-phospho-ChK1S296 (clone D3O9 F, Cell signaling Technology, cat #90,178) and total ChK1 (clone 2G1D5, Cell signaling Technology, cat #2360).

Techniques:

Fig. 2 Notch1 blockade affects histone expression. A Bubble plot depicting the most significantly inhibited pathways in K457 melanoma cells treated with 50ug/ml anti-Notch1 for 48 h. B Heat Map depicting histone mRNAs significantly downregulated in the cells from A. C-D Core histone protein levels in K457 (C) and A375 (D) cells treated as in A. α-tubulin was used as loading control. Band intensity is the mean between two experimental repeats for each cell line, normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 treated cells normalized to ctrl

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 2 Notch1 blockade affects histone expression. A Bubble plot depicting the most significantly inhibited pathways in K457 melanoma cells treated with 50ug/ml anti-Notch1 for 48 h. B Heat Map depicting histone mRNAs significantly downregulated in the cells from A. C-D Core histone protein levels in K457 (C) and A375 (D) cells treated as in A. α-tubulin was used as loading control. Band intensity is the mean between two experimental repeats for each cell line, normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 treated cells normalized to ctrl

Techniques: Expressing, Control

Fig. 3 Notch1 blockade causes replication stress and DNA damage. A % of K457 cells in each phase of the cell cycle at 48 h treatment with anti-N1 (100ug/ml). B DNA replication fork speed in K457 cells. 100 fibers were analyzed for each group. Speed: IdU (red) tract length/time of the IdU pulse (30 min) = um/min. um were converted to Kilobases (Kb) using the conversion factor 1um = 2.59 kb. C pChK1S345 is induced because of RF stress and ATR activation. Band intensity is the mean between two experimental repeats normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 treated cells normalized to ctrl. D γH2 AX foci in K457 cells treated as in A. n ≥ 50 nuclei per section were counted for 5 section per group. E Neutral Comet Assay of K457 cells treated as in A. n ≥ 200 comets/group. Significance of the differences among groups was determined by the Student’s t test. Data are the mean of three independent experiments each performed in triplicate

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 3 Notch1 blockade causes replication stress and DNA damage. A % of K457 cells in each phase of the cell cycle at 48 h treatment with anti-N1 (100ug/ml). B DNA replication fork speed in K457 cells. 100 fibers were analyzed for each group. Speed: IdU (red) tract length/time of the IdU pulse (30 min) = um/min. um were converted to Kilobases (Kb) using the conversion factor 1um = 2.59 kb. C pChK1S345 is induced because of RF stress and ATR activation. Band intensity is the mean between two experimental repeats normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 treated cells normalized to ctrl. D γH2 AX foci in K457 cells treated as in A. n ≥ 50 nuclei per section were counted for 5 section per group. E Neutral Comet Assay of K457 cells treated as in A. n ≥ 200 comets/group. Significance of the differences among groups was determined by the Student’s t test. Data are the mean of three independent experiments each performed in triplicate

Techniques: Activation Assay, Control, Neutral Comet Assay

Fig. 4 Concurrent blockade of Notch1 and ChK1 increases replication stress and DNA damage. A pChK1S345, pChK1S296 (autophosphorylation site that leads to full activity) and total ChK1 in K457 cells treated with anti-N1 (100ug/ml) and prexasertib (prex) (5 nM), alone or in combination, for 48 h. Band intensity is the mean between two experimental repeats normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 and prex treated cells normalized to ctrl. B DNA replication fork speed. 100 fibers were analyzed for each group. Speed: IdU (red) tract length/time of the IdU pulse (30 min) = um/min. um were converted to Kilobases (Kb) using the conversion factor 1um = 2.59 kb.). Representative DNA tracts are shown on the right. C % of K457 cells treated as in A, in each phase of the cell cycle. Significance between treatment for each phase was calculated by the Student’s t test (table below). D γH2 AX foci in cells treated as in A. n ≥ 50 nuclei per section, 5 section per group. Representative pictures are shown below. E Neutral Comet Assay of cells treated as in A. n ≥ 200 comets/group. Representative comets are shown below. Data are the mean of three independent experiments each performed in triplicate. Significance of the differences among groups was determined by the Student’s t test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 4 Concurrent blockade of Notch1 and ChK1 increases replication stress and DNA damage. A pChK1S345, pChK1S296 (autophosphorylation site that leads to full activity) and total ChK1 in K457 cells treated with anti-N1 (100ug/ml) and prexasertib (prex) (5 nM), alone or in combination, for 48 h. Band intensity is the mean between two experimental repeats normalized to the loading control for each lane. Control was then set at 1 and the levels in aN1 and prex treated cells normalized to ctrl. B DNA replication fork speed. 100 fibers were analyzed for each group. Speed: IdU (red) tract length/time of the IdU pulse (30 min) = um/min. um were converted to Kilobases (Kb) using the conversion factor 1um = 2.59 kb.). Representative DNA tracts are shown on the right. C % of K457 cells treated as in A, in each phase of the cell cycle. Significance between treatment for each phase was calculated by the Student’s t test (table below). D γH2 AX foci in cells treated as in A. n ≥ 50 nuclei per section, 5 section per group. Representative pictures are shown below. E Neutral Comet Assay of cells treated as in A. n ≥ 200 comets/group. Representative comets are shown below. Data are the mean of three independent experiments each performed in triplicate. Significance of the differences among groups was determined by the Student’s t test

Techniques: Activity Assay, Control, Neutral Comet Assay

Fig. 5 Concurrent blockade of Notch1 and ChK1 causes mitotic catastrophe and cell death. A % of A375 cells positive for cleaved-caspase-3 (apoptotic), KI67 (proliferative) or double positive (mitotic catastrophe) after treatment with anti-N1 (100ug/ml) and prexasertib (5 nM), alone or in combination, for 24 h. Representative staining of each condition is shown on the right (scale bar: 10um). B Survival of A375 at 48 h post treatment determined by trypan blue staining. C Clonogenic assay of A375 treated with anti-N1, prex and a combination of the two. A picture of representative colonies for each condition is shown on the right. D normal human melanocytes and human fibroblasts treated as the cells in B. Statistical differences were determined by the Student’s t test. Data are the mean of three independent experiments each performed in triplicate

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 5 Concurrent blockade of Notch1 and ChK1 causes mitotic catastrophe and cell death. A % of A375 cells positive for cleaved-caspase-3 (apoptotic), KI67 (proliferative) or double positive (mitotic catastrophe) after treatment with anti-N1 (100ug/ml) and prexasertib (5 nM), alone or in combination, for 24 h. Representative staining of each condition is shown on the right (scale bar: 10um). B Survival of A375 at 48 h post treatment determined by trypan blue staining. C Clonogenic assay of A375 treated with anti-N1, prex and a combination of the two. A picture of representative colonies for each condition is shown on the right. D normal human melanocytes and human fibroblasts treated as the cells in B. Statistical differences were determined by the Student’s t test. Data are the mean of three independent experiments each performed in triplicate

Techniques: Staining, Clonogenic Assay

Fig. 6 Concurrent blockade of Notch1 and ChK1 improves the survival of animals bearing MBMs. A 105 A375 cells expressing luciferase, were inoculated stereotactically into the right cerebral cortex of athymic nude mice. Tumors were monitored twice weekly since inoculation. Kaplan–Meier curves were generated with Graph Pad Prism10. Logrank p values, median overall survival (OS) and Hazard Ratio (HR) are shown. B Representative H&E staining. Scale bar: 200um

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

doi: 10.1186/s13046-025-03411-w

Figure Lengend Snippet: Fig. 6 Concurrent blockade of Notch1 and ChK1 improves the survival of animals bearing MBMs. A 105 A375 cells expressing luciferase, were inoculated stereotactically into the right cerebral cortex of athymic nude mice. Tumors were monitored twice weekly since inoculation. Kaplan–Meier curves were generated with Graph Pad Prism10. Logrank p values, median overall survival (OS) and Hazard Ratio (HR) are shown. B Representative H&E staining. Scale bar: 200um

Techniques: Expressing, Luciferase, Generated, Staining

Inhibition of the Notch pathway aggravates DSS-induced colitis. CT: control, LY411,575 (Notch inhibitor): 10 mg/kg LY411,575 gavage, DSS: 2.5% DSS water, DSS-LY411,575: LY411,575 was intragastric administrated at the last 4 days of DSS feeding (once per day). (A) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (B) Serum FD-4 concentration in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (C) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (D) Representative colonic H&E staining image of each group (scale bar = 50 μm). (E) Representative colonic IF staining image of claudin-1 ( green ) and claudin-3 ( green ) (scale bar = 25 μm). (F) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) (scale bar = 25 μm). Quantification of N1ICD and Hes1 were determined by the ratio of positive staining cells per crypt. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Inhibition of the Notch pathway aggravates DSS-induced colitis. CT: control, LY411,575 (Notch inhibitor): 10 mg/kg LY411,575 gavage, DSS: 2.5% DSS water, DSS-LY411,575: LY411,575 was intragastric administrated at the last 4 days of DSS feeding (once per day). (A) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (B) Serum FD-4 concentration in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (C) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (D) Representative colonic H&E staining image of each group (scale bar = 50 μm). (E) Representative colonic IF staining image of claudin-1 ( green ) and claudin-3 ( green ) (scale bar = 25 μm). (F) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) (scale bar = 25 μm). Quantification of N1ICD and Hes1 were determined by the ratio of positive staining cells per crypt. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Inhibition, Control, Concentration Assay, Staining

Notch pathway is inhibited in the colon under VDd or VDR KO conditions. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO (A) and VDd (B) mice (scale bar = 25 μm). (C, D) Real-time PCR analysis of the gene expression of Notch ligands, receptors, and effectors in the colon in VDR KO (C) and VDd (D) mice. Expression levels (VDR KO vs. WT or VDd vs. CT) were compared using paired t -test. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Notch pathway is inhibited in the colon under VDd or VDR KO conditions. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO (A) and VDd (B) mice (scale bar = 25 μm). (C, D) Real-time PCR analysis of the gene expression of Notch ligands, receptors, and effectors in the colon in VDR KO (C) and VDd (D) mice. Expression levels (VDR KO vs. WT or VDd vs. CT) were compared using paired t -test. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

VD/VDR modulates the Notch pathway in colitis. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDd-DSS and respective control colitis mice (scale bar = 25 μm). (C, D) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO-DSS and respective control colitis mice. To activate VDR, mice were administered paricalcitol (PAR) 7 days before drinking DSS water and were sustained until sacrifice. (E) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (F) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (G) Representative colonic H&E staining image of each group (scale bar = 50 μm). (H) Representative colonic IF staining image of claudin-1 ( green ), claudin-3 ( green ), and Hes1 ( red ) (scale bar = 25 μm). Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR modulates the Notch pathway in colitis. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDd-DSS and respective control colitis mice (scale bar = 25 μm). (C, D) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO-DSS and respective control colitis mice. To activate VDR, mice were administered paricalcitol (PAR) 7 days before drinking DSS water and were sustained until sacrifice. (E) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (F) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (G) Representative colonic H&E staining image of each group (scale bar = 50 μm). (H) Representative colonic IF staining image of claudin-1 ( green ), claudin-3 ( green ), and Hes1 ( red ) (scale bar = 25 μm). Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Techniques: Staining, Control

VD/VDR signaling regulates the Notch pathway and TJs in Caco-2 cells. TNF-α (100 ng/mL) was added to mimic inflammatory stimulation, and 100 nM PAR was used to activate VDR; the solvate was added as control (CT). Specific siRNA of VDR (SiVDR) was used to downregulate VDR, negative control (NC) sequence was used as control (scale bar = 10 μm). (A) Representative IF staining image of claudin-1 ( green ) and claudin-3 ( green ) in Caco-2 cells treated with or without PAR before TNF-α intervention. (B) Representative IF staining image of N1ICD ( red ) and Hes1 ( red ) in Caco-2 cells with or without VDR downregulation before TNF-α intervention. (C) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in Caco-2 cells after VDR downregulation.

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling regulates the Notch pathway and TJs in Caco-2 cells. TNF-α (100 ng/mL) was added to mimic inflammatory stimulation, and 100 nM PAR was used to activate VDR; the solvate was added as control (CT). Specific siRNA of VDR (SiVDR) was used to downregulate VDR, negative control (NC) sequence was used as control (scale bar = 10 μm). (A) Representative IF staining image of claudin-1 ( green ) and claudin-3 ( green ) in Caco-2 cells treated with or without PAR before TNF-α intervention. (B) Representative IF staining image of N1ICD ( red ) and Hes1 ( red ) in Caco-2 cells with or without VDR downregulation before TNF-α intervention. (C) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in Caco-2 cells after VDR downregulation.

Techniques: Control, Negative Control, Sequencing, Staining

VD/VDR-mediated TJ protection is partly dependent on the Notch pathway. LY411,575 (1 μM) was used to inhibit the Notch pathway in Caco-2 cells, and 100 nM PAR was used to activate VDR. The solvate was added as control (CT). Representative IF staining image of N1ICD ( red ), Hes1 ( red ), claudin-1 ( green ), and claudin-3 ( green ) in Caco-2 cells treated with or without LY411,575 before PAR intervention (scale bar = 10 μm).

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR-mediated TJ protection is partly dependent on the Notch pathway. LY411,575 (1 μM) was used to inhibit the Notch pathway in Caco-2 cells, and 100 nM PAR was used to activate VDR. The solvate was added as control (CT). Representative IF staining image of N1ICD ( red ), Hes1 ( red ), claudin-1 ( green ), and claudin-3 ( green ) in Caco-2 cells treated with or without LY411,575 before PAR intervention (scale bar = 10 μm).

Techniques: Control, Staining

VD/VDR signaling positively regulates the Notch-1/Hes1 pathway. (A) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in cultured intestinal organoids (scale bar = 10 μm). (B) Western blot analysis of N1ICD and Hes1 proteins in cultured intestinal organoids from WT and VDR KO mice. (C) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in cultured intestinal organoids from WT and VDR KO mice. (D) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after TNF-α intervention. (E, F) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after downregulation of VDR with siVDR (E) or activation of VDR with PAR (F) . Data are presented as the mean ± SEM from three separate experiments, the statistical analysis performed with paired t -test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling positively regulates the Notch-1/Hes1 pathway. (A) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in cultured intestinal organoids (scale bar = 10 μm). (B) Western blot analysis of N1ICD and Hes1 proteins in cultured intestinal organoids from WT and VDR KO mice. (C) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in cultured intestinal organoids from WT and VDR KO mice. (D) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after TNF-α intervention. (E, F) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after downregulation of VDR with siVDR (E) or activation of VDR with PAR (F) . Data are presented as the mean ± SEM from three separate experiments, the statistical analysis performed with paired t -test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Techniques: Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activation Assay

VD/VDR positively regulates Notch-1 transcription. (A) Western blot analysis of VDR, Notch-1, N1ICD, and Hes1 in Caco-2 cells after siVDR and PAR intervention. (B) Graphs of the predicted VDR binding region in the Notch-1 promoter. (C) Dual-luciferase reporter assay of VDR to Notch-1 promoter. Notch-1 promoter (Notch-1) and negative control (CON) were constructed in a GV238 vector. The VDR cDNA and vector (pcDNA3.1) were linked to upregulate VDR (pVDR). Data are presented as the mean ± SEM from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR positively regulates Notch-1 transcription. (A) Western blot analysis of VDR, Notch-1, N1ICD, and Hes1 in Caco-2 cells after siVDR and PAR intervention. (B) Graphs of the predicted VDR binding region in the Notch-1 promoter. (C) Dual-luciferase reporter assay of VDR to Notch-1 promoter. Notch-1 promoter (Notch-1) and negative control (CON) were constructed in a GV238 vector. The VDR cDNA and vector (pcDNA3.1) were linked to upregulate VDR (pVDR). Data are presented as the mean ± SEM from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Techniques: Western Blot, Binding Assay, Luciferase, Reporter Assay, Negative Control, Construct, Plasmid Preparation

Review

cleaved notch 1 (Cell Signaling Technology Inc)

Structured Review

Images

1) Product Images from "Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin"

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